ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis

The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biology. Synthetic cellular systems are envisioned as out-of-equilibrium enzymatic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metabolism. Importantly, gaining tight control over the external medium is essential to avoid thermodynamic equilibrium due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymatic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable l-arginine breakdown. In addition, we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium composition and to achieve sustainable glycerol 3-phosphate synthesis.


A) glpK
Table S1 | Primers used to clone glpK into pRSETA and percevalHR into pBAD24. The primers contain an uracil base required for uracil excision 1 .   We measured the osmolality of the solution encapsulated in vesicles with a freezing point osmometer (Osmomat 3000 basic, Gonotec). We included the encapsulated components (1 µM ArcA, 2 µM ArcB, 5.8 µM PercevalHR, 10 mM Na-ADP, 10 mM MgCl 2 , 0.5 mM L-ornithine) without pre-formed proteoliposomes (replaced with 50 mM KPi) and without ArcC, stored in 10% vol/vol glycerol (replaced with 50 mM KPi plus 100 mM KCl, under the assumption that glycerol is rapidly diluted out and that ArcC does not significantly contribute to osmolality). We measured an osmolality value of 227 mOsm/kg. We later stored all enzymes in 10% vol/vol glycerol, but since we did not assume a glycerol contribution to the osmolality, we did not repeat the measurement. We prepared a calibration curve of 50 mM KPi pH 7.0 with increasing concentrations of NaCl. From the linear fit (slope = 1.928 ± 0.02711; y(x0) = 114.9 ± 1.443), we found that 58 mM NaCl in 50 mM KPi pH 7.0 is required to match the internal osmolality.

Figure S5 | Estimation of internal nucleotide concentration in the chemiluminescence assay.
We assume an average specific internal volume of 2.7 µL/mg total lipids (18 µL per 6.66 mg total lipids) 2 . For ATP and total nucleotide quantification, samples of 100 µL of 2.7 mg/mL total lipids are diluted out to 160 µL, yielding 1.6875 mg/mL due to PCA and KOH/KHCO 3 addition. From this, a volume of 60 µL is used for ATP or total nucleotide determination, consisting of 0.1 mg total lipids and an in internal volume of 0.27 µL (0.45% vol/vol). The chemiluminescence assay yields 3 nmol of nucleotides, corresponding to an internal concentration 11.1 mM, which is in good agreement with the encapsulated concentration of 10 mM of ADP (given the uncertainty in the estimation of the specific internal volume of liposomes).

Figure S6 | Estimation of internal glycerol concentration.
We calculate that samples of 120 uL and a total lipid concentration of 2.7 mg/mL (used for the online measurement of ATP/ADP) consist of 0.324 mg of lipids and an internal volume of 0.8748 uL (0.73% vol/vol). Thus, we apply a correction factor of 137 to determine the internal glycerol concentration (total internal volume, black slope). This correction factor may be further adjusted by taking into account that the "active" volume, i.e. the volume that contains all components and leads to a functional pathway, is smaller that the total internal volume due to all components not being present in all liposomes 6-8 . From the chemiluminescence experiments (that gave a 50% conversion, see Figure2e in the main text), we estimate an active volume of 0.5x the total internal volume, leading to a correction factor of 274 (active internal volume, red slope). In the glycerol titration experiment, 20-50 mM glycerol was cumulatively added to the samples (blue and green thick lines; dashed lines are single additions; see main figure), which is well in excess of the 10 mM ADP initially present. The correction factor of 274 also holds for the chemiluminescence experiments.

Figure S7 | Calibration curve of PercevalHR in solution and in liposomes.
A calibration curve was prepared for PercevalHR in solution (black dots; n=2 and error bars represent s.d.) and in vesicles (red dots; n=2 and error bars represent s.d.). The in solution calibration curve was obtained by calculating the F500/F430 ratio from spectra acquired in the presence of different ATP/ADP ratios (with ATP plus ADP always equal to 10 mM). The curve was fitted with a Hill equation (n fixed to 1) and found to be in good agreement with previously reported curves 2 . The vesicle samples containing 1:9, 1:1 and 9:1 ATP/ADP (ATP plus ADP equal to 10 mM) were found to significantly deviate from the calibration curve in solution. Therefore, we report the F500/F430 values rather than ATP/ADP ratios.  A control that the proton and potassium gradients are fully dissipated and do not affect the PercevalHR readout by altering the pH is taken by using a ten-fold excess of the ionophores nigericin and valinomycin. The vesicle composition was as reported in Figure 2a in the Main text, with the exception that nigericin and valinomycin were added to a final concentration of 10 µM (black trace; gives final 3.6% vol/vol DMSO) and that no glycerol was present. To control for undesired side effects due to the higher DMSO concentration, a control was taken with the standard ionophore concentration of 1 µM (red trace; gives final 0.4% vol/vol DMSO), to which 3.6% vol/vol DMSO was additionally added. We find that the ATP/ADP levels are comparable with 1 µM and 10 µM nigericin and valinomycin. In addition, the higher DMSO concentration did not have a significant effect on the vesicle stability.
Figure S10 | pH of external medium over time. The external pH was measured with a pH electrode over time in 500 µL 2.7 mg/mL of vesicles to which 5 mM L-arginine was added. The vesicle composition was as reported in Figure 2b in the Main text, with the exception that no glycerol was used.